Analysis of Oxidative Damage by Gene‐Specific Quantitative PCR

This unit describes the gene-specific quantitative PCR-based (QPCR) assay, which is used to measure DNA integrity of both nuclear and mitochondrial genomes based on amplification of long DNA targets. QPCR can be used to quantify the formation of DNA damage and the kinetics of DNA repair by following restoration of amplification of the target DNA over time after removal of the damaging agent. A detailed protocol to set up QPCR in any laboratory, highlighting critical parameters for successful establishment of the assay and interpretation of the results, is provided here. Advantages (e.g., the use of nanogram amounts of DNA) and limitations (e.g., the inability to define the specific type of lesion present on the DNA) of using QPCR to assay DNA damage in human cells are also described. Curr. Protoc. Hum. Genet. 62:19.1.1-19.1.13. © 2009 by John Wiley & Sons, Inc. Keywords: mitochondrial and nuclear DNA integrity; oxidative stress; reactive oxygen species; DNA damage and repair; QPCR